General Protocols for Preparation of Plasmid DNA Template, RNA In Vitro Transcription, and RNA Purification by Denaturing PAGE

The development of methods for in vitro transcription of defined RNA sequences has been a key factor driving the tremendous advances in RNA biology over the last three decades. The numerous approaches available today to study RNA structure and function vary widely in their demands on the quality and quantity of material needed. These range for example from a few micrograms in biochemical assays, RNA structure probing or RNA folding studies using UV melting, to up to tens of milligrams or more of highly purified RNA for structural studies by nuclear magnetic resonance (NMR) or X-ray crystallography. Therefore, robust and scalable protocols, such as those described in this chapter, for production of plasmid DNA template, RNA in vitro transcription, and RNA purification, are an essential component of any RNA laboratory’s experimental repertoire.